Holmgren, Benjamin T.

Abstract [en]

RNA interference (RNAi) is a gene regulation mechanism conserved among eukaryotes. To silence gene expression, RNAi relies on a short single-stranded guide RNA to steer the RNA-induced Silencing Complex (RISC) to mRNAs with guide strand-complementary sequences. RNAi is a highly membrane-associated process. The RISC complex is likely loaded at the rough Endoplasmic Reticulum, where it can bind to and degrade mRNAs. Components of the RISC complex also colocalize to late endosomes, and the efficiency of RNAi-mediated silencing is affected by changes in late endosome to lysosome fusion. RNAi can be systemic and inherited, effecting gene silencing in distal tissues and in the offspring.

In this thesis, the model organism Caenorhabditis elegans was used to identify and characterize factors connecting systemic and inherited RNAi to the endomembrane system. We identify two SNARE proteins, SEC-22 and SYX-6, that both act as negative regulators of RNAi. SNAREs are necessary for vesicle fusion. Both SEC-22 and SYX-6 localize to late endosomes, and both interact with systemic RNAi protein SID-5 in a yeast two-hybrid (Y2H) screen. We find that in addition to its function in systemic RNAi, SID-5 is required for proper maturation of late endosomes. Furthermore, we identify the putative RNA-binding protein C12D8.1 as a novel regulator of RNAi inheritance. Mutant C12D8.1 animals will have enhanced inheritance of RNAi silencing, which negatively affects the ability of the progeny to silence new targets using RNAi. Finally, we describe a novel, object-based method for estimating significance in colocalization studies. This method helped us describe and quantify spatial relations between fluorophore-labeled proteins in situations where such analyses would otherwise be impossible.

In conclusion, the work presented here further elucidates the connection between cellular RNAi, the endomembrane system, and the outside world.

Zhao, Yani

Abstract [en]

The membrane system of a eukaryotic cell is a large and complex system handling the transport, exchange and degradation of many kinds of material. Recent research shows that double-stranded RNA (dsRNA) mediated gene silencing (RNA interference) is a membrane related process. After long dsRNA is processed to small interfering RNA (siRNA) by Dicer, the guide strand and passenger strand are separated in the RNA induced silencing complex (RISC) by Argonaute. The process of loading siRNA into RISC has been suggested to occur at the rough Endoplasmic Reticulum (rER).The components of RISC also associate with late endosomes/multivesicular bodies (MVBs). Furthermore, disturbing the balance between late endosomes/MVBs and lysosomes has been shown to affect the efficiency of silencing.

We use the nematode Caenorhabditis elegans as our model organism to study two questions: how does membrane transport affect RNAi and spreading of RNAi from the recipient cells to other tissues (systemic RNAi); and how does RNA transport contribute to the multigenerational silencing induced by dsRNA (RNAi inheritance)? Using SID-5, a protein required for efficient systemic RNAi, as bait in a yeast two-hybrid (Y2H) screen, we got 32 SID-5 interacting candidate proteins. Two of these are the SNARE protein SEC-22 and the putative RNA binding protein C12D8.1. In two additional Y2H screens, we found that SID-5 interacts with multiple syntaxin SNAREs, including SYX-6, whereas SEC-22 only interacts with SYX-6. SNAREs usually function in vesicle fusion processes. We found the two SNARE proteins SEC-22 and SYX-6 to be negative regulators of RNAi and to localize to late endosomes/MVBs. In addition, loss of sid-5 leads to an endosome maturation defect. Finally, we found that the putative RNA binding protein C12D8.1 negatively regulates RNAi inheritance and that C12D8.1 mutant animals show impaired RNAi upon targeting a new gene. Taken together, the results presented in this thesis provide us with more evidence for the connection of the membrane transport system and RNAi. The identification of a putative negative regulator of RNAi inheritance further enriches this research field.